Adaptation to anaerobiosis is a complex physiological response demonstrated by facultative bacteria such as Escherichia coli. One important aspect of this adaptation is the ability to respire alternate terminal electron acceptors in place of oxygen. Nitrate is an important alternate electron acceptor, and synthesis of the respiratory enzyme, nitrate reductase, is induced by nitrate in the absence of oxygen. In addition, functioning nitrate respiration inhibits the formation of other facultative respiratory and fermentation pathways. The long-range goal of this project is to identify and characterize regulatory elements and networks that control the formation of nitrate reductase and other facultative respiratory enzymes. This proposal describes a molecular genetic approach toward understanding the control of nitrate respiration. It focuses on the nar (ch1C) locus, which contains both structural genes and regulatory genes for nitrate reductase. The nar locus will be examined in detail, combining deletion mapping, complementation analysis, molecular cloning and examination of gene-protein relationships. A collection of nar point, insertion and deletion mutations will be isolated by a variety of methods and screening procedures. These mutations will be used to construct a detailed fine-structure genetic map of the nar region. Selected mutations will be tested for genetic complementation, allowing division of the fine-structure map into complementation groups. The nar region will be cloned and analyzed by restriction mapping and Southern blot analysis. These studies will allow the correlation of the genetic fine-structure, complementation and physical maps, and will serve to precisely locate the genes of the nar region. The structural genes for nitrate reductase will be identified by testing various mutants and subclones for production of nitrate reductase polypeptides. Finally, operon fusions will be used to analyze the regulation of nitrate reductase and other facultative respiratory genes, and also to test the effects of specific mutations in nar regulatory genes.